The invention relates to a method for monitoring a cryopreserved biological sample, in particular a method for controlling the state of a cryopreserved biological sample. Furthermore, the invention relates to a monitoring device, which is configured for monitoring a cryopreserved biological sample, in particular according to the said method. Applications of the invention are given in the state and quality control of cryopreserved samples, in particular in long-term storage (e.g. in banks with biological samples) or in the clinical sample control.
Cryopreservation is the only known technology, which has proven suitable for a life-sustaining and/or function-preserving long-term storage or archiving of biological samples, such as biological cells (including gametes), cell constituents, cell compositions, tissues or tissue parts, under practical conditions. Based on the most recent development in the reproductive and regenerative medicine, in the so-called “Tissue Engineering” and in cellular biology (in particular stem cell biology) and due to new applications of cryopreservation, for example in the protection of species, a strong demand exists for massive-scale storage of biological samples in the cryopreserved state.
One important requirement towards cryopreservation of biological samples consists in the fact that the samples are to be stored free of damages. The application of biological samples after cryopreservation, for example during implantation of living biological cells into an organism, can result in dramatic sanitary consequences for the organism in case the samples have suffered for example genetic, epigenetic or functional damage during cryopreservation. Damages can occur in particular through changes in the sample constituents, such as aqueous sample constituents, during cooling down or thawing of the sample. Whilst the steps of cooling down or of thawing of the biological samples are examined for possible damaging potentials and optimized for the practical application, experiences on possible changes of the samples in the cryopreserved state have been only limitedly available to date. Checking the sample quality is at this point in time possible only by partial or complete thawing of the sample. Interim thawing of a cryopreserved sample, however, increases the risk of damage.
To date, it is assumed that below the glass transition temperature of water at −137° C. any chemical conversion is slowed down in a practically infinite manner and that no recrystallization effect can occur in this temperature range of water. Therefore, a sample, which was led damage-free in the temperature range below −130° C. is considered stably preserved. This assumptions are made in particular for the vitrification of biological samples, wherein the sample is frozen with such high cooling rate that no damaging ice crystals can be formed.
It is known from the practice, however, that, even if the formation of ice crystals is avoided during cooling down or thawing, the samples can exhibit damages after the thawing. To date, however, monitoring of the state or the quality of long-term stored samples does not take place. Even if there is the option of taking a partial sample from a diversity of samples and, after their thawing, analyzing their state, it is, however, not possible to draw conclusions from a partial sample for other partial samples or for the bulk sample due to the statistical nature of the damaging processes that take place. Reliable technical solutions for monitoring the state of the samples in situ, thus in the deep-cold state, are not available to date.
WO 2006/058816 discloses a sample container for a cryopreserved sample, wherein an optical analysis of the sample is provided for in the sample container. It is suggested to detect recrystallization processes through reflexion measurements or transmission measurements. The measurements are, however, not suitable for a quantitative sample characterization. They are only limitedly applicable even for qualitative observations, since the measured light intensities not only depend on a possible sample-recrystallization, but also sensitively on other effects. It is furthermore suggested that the sample is provided with a fluorescence marker (fluorescence staining), in order to monitor the formation of extra-cellular ice, wherein, however, no statement is made on the detection of a crystallization state of the sample.
A further sensitive method of analysis is the generally known Raman spectroscopy. This method is, for example, mentioned in US 2006/0082762 A1 or by R. S. Hawke et al. (in “Rev. Sci. Instr.” Vol. 45, 1974, p. 1598 ff.) without, however, addressing the measurement on biological samples. It is suggested in US 2004/0073120 A1 to perform Raman spectroscopic measurements on biological tissue samples. The measurements can, however, only be performed on samples at room temperature. Raman spectroscopic measurements on biological samples at room temperature are also mentioned in EP 1 438 421 B1, DE 101 23 443 A1, DE 36 87 438 T2, US 2008/0306346 A1 and US 2006/0155195 A1.
It is known from the publication of C. A. Tulk et al. in “Journal of Chemical Physics” (Vol. 109, 1998, p. 8478) to characterize differences of the chemical linkage in amorphous ice and crystalline ice through Raman spectroscopic measurements. Application of these measurements, which were performed with specialized spectroscopic apparatuses for the fundamental research, are not mentioned by C. A. Tulk et al.
The objective of the invention is to provide an improved method for monitoring a cryopreserved biological sample by means of which disadvantages of conventional techniques are overcome. The monitoring method should in particular allow sample observation without any change on the sample and/or gaining of quantitative information about the crystallization state (in particular the degree of crystallization) and/or about chemical changes in the sample with improved reliability and/or accuracy. The objective of the invention is furthermore to provide an improved device for monitoring a cryopreserved biological sample by means of which disadvantages of conventional techniques are overcome. The monitoring apparatus should in particular be compatible with available techniques for cryopreservation of biological samples.
These objectives are achieved by a method and a device, respectively, of the invention.